Aims: To gain new insights into the metabolic contribution of bacterial group in the rumen. Methods and Results: Both DNA- and RNA-derived bacterial 16S ribosomal materials from bovine rumen contents were used as the template for bacterial community and analyse microbiota by three methods namely custom phylogenetic microarray, quantitative real-time PCR and denaturing gradient gel electrophoresis techniques. Bacterial analysis showed that genera affiliating with the Proteobacteria apparently made a greater metabolic contribution to rumen function than their population sizes indicated. Analysis of another rumen microbial group, the methanogens, using clone libraries for the expressed methyl coenzyme reductase subunit A (mcrA) revealed that an uncultivated methanogen clade contributes one-third of RNA-derived mcrA sequences based on a limited number of clones analysed. These uncultivated methanogen species were not observed in the mcrA gene library based on the DNA-derived sequences. Conclusions: The comparison of results obtained from DNA- and RNA-derived materials suggests that some of the Proteobacteria and novel methanogen species appeared to be low in abundance in the rumen maintained on grain-based diets might play a greater role in rumen metabolism. Significance and Impact of the Study: These studies provide the first report to compare high-throughput analysis of bacterial 16S rRNA genes from DNA- and RNA-derived materials to indicate differences that species make to community structure and metabolic activity.